MIP75ROI

// -Written in IJ1 Macro Fiji-

//@File (label = "Input directory", style = "directory") chosenInputDir
//@File (label = "Output directory", style = "directory") chosenOutputDir
//@String (label = "File suffix", value = ".lif") fileExtension


// Macro for measuring 0.75 times smaller objects within original objects
// This macro runs on all imaging formats, batch process image by image (designed for lif series).
// Macro written by alina.gavrilov@roche.com (with great help from jakob.stueber@roche.com and BioVoxxel (Jan Brocher)) 04.2020
// Suggestions for improvement and constructive criticism are more than welcomed.

// This Macro will:
// 1) Get information from user about experiment to run analysis, get input and output directory for batch
// 2) Generate Z-stack projection and MIP projection for all channels
// 3) Create a mask on desired channel (called "DAPI") with Triangle threshold method + create ROI(s)
// 4) Reduce the ROI(s) to 75% and place a new ROI in the center of detected object(s)
// 5) Overlay selected ROI(s) to channel of interest (called "ChOI")
// 6) Measure the previously set parameters within 75% object(s) + create results table
// 7) Save tifs of all images as merged channels and overlayed ROI(s) on channel of interest and mask  
// 8) Export measurements as excel and as txt files

// Show info which directories user selected
print ("Your selected input directory is " + chosenInputDir + ".");
print ("Your selected input directory is " + chosenOutputDir + ".");

// 1) Get information from user about experiment to run analysis, get input and output directory for batch
// Dialogue: Select input and output directory and file format (only tested with lif!) --> 3 strings on top of macro
// Dialogue: Which channel is mask channel (=DAPI) and channel of interest to be measured (=ChOI)? 
Dialog.create("DAPI/mask channel selection");  
Dialog.addChoice("Which channel is your DAPI/mask channel?", newArray("1","2","3","4","5"));
Dialog.show();
   DAPI = Dialog.getChoice();
   print ("Your selected DAPI/mask is channel " + DAPI + ".");

Dialog.create("Channel of interest selection");  
Dialog.addChoice("What channel do you want to measure (channel of interest)?", newArray("1","2","3","4","5"));
Dialog.show();
   ChOI = Dialog.getChoice();
   print ("Your selected channel of interest is channel " + ChOI + ".");

// NOT DONE - Open first image of experiment, split channels
// NOT DONE - Only if you got time: Adjust Auto-Brightness/Contrast for each channel (count channels, somehow with loop)
// NOT DONE - Rename user defined DAPI channel on opened image (or even place a bold "this is mask channel" on top)
// NOT DONE - Rename user defined Channel of interest on opened image (only for visualization for user + confirmation) 

// Dialogue/boolean: Are you certain that this is right? Yes/No/Cancel
// Answers: Yes --> Hint "Your analysis has started" + run script ; No --> "Well, try again..." + abort script, close all; Cancel --> abort script, close all

if (getBoolean("Your DAPI/mask is channel " + DAPI + " and your channel of interest to be measured is channel " + ChOI + ". Is this correct?")) {   
	showMessageWithCancel("Great! Your analysis will start after pressing Ok. Afterwards there is no way out ;). Open images & results will be closed.");
//	showMessage("Great! Your analysis has started. There is no way out ;). Open images & results will be closed.");
	}
else
	{
	run("Close All");
	exit("Well. I suggest, you get a coffee, think about your experiment and try again later. Analysis will be not performed.");    // stops script 
	};

// NOT DONE - Close all open images without saving (NOT DONE - only saves threshold informations for tiff files later on)
// All following procedures will be done on ONE image. This will be analyzed, saved, closed. Only then, next one opens.
run("Close All");
// Closes the "Results" window
    if (isOpen("Results")) {
         selectWindow("Results"); 
         run("Close" );
    }

// 2) Generate Z-stack projection and MIP projection for all channels
// Batching intro here --> will open one image of a series, analyse, close, open next etc.
inputDir = chosenInputDir + File.separator;
outputDir = chosenOutputDir + File.separator;

run("Bio-Formats Macro Extensions");

processBioFormatFiles(inputDir); 	// This guy runs to whole script coming below.

// Save as excel and Save as txt files to output directory 
saveAs("Results", outputDir + "/all_results.csv");
saveAs("Results", outputDir + "/all_results.txt");

exit("Done! Your analysis is completed and your results are saved to " + outputDir + " .");

function processBioFormatFiles(currentDirectory) {

	fileList = getFileList(currentDirectory);

	for (file = 0; file < fileList.length; file++) {

		if (endsWith(fileList[file], fileExtension)) {
			Ext.setId(currentDirectory + fileList[file]);
			Ext.getSeriesCount(seriesCount);
			
			for (series = 1; series <= seriesCount; series++) {
				//record the Bio-Formats importer with the setup you need if different from below and change accordingly
// Bio-Format importer: Hyperstack, Composite
				run("Bio-Formats Importer", "open=[" + currentDirectory + fileList[file] + "] autoscale color_mode=Composite display_rois rois_import=[ROI manager] view=Hyperstack stack_order=XYCZT series_"+series);
				//add your macro code here
	print("Processing: " + getTitle() + series);

// Z-projection of all stacks with MIP projection for each channel in composite mode
	title = getTitle();
	new_title = "MAX_" + title;
	selectWindow(title);	
	run("Z Project...", "projection=[Max Intensity]");
	
// Close original image, only work with MAX_... image
	selectWindow(title);	
	close();
	
// Save tif image of composite view
// Duplicate MAX_ image
	run("Duplicate...", "duplicate");
	
// Add scale bar // Feel free to change predifined parameters
	run("Scale Bar...", "width=100 height=6 font=24 color=White background=None location=[Lower Right] bold overlay");

// Convert merged image to RGB format with Flatten function
	run("Flatten");

// Save as tif with suffix _merged
	saving_title = replace(new_title, "/", "_" );
	saveAs("Tiff", outputDir + saving_title + "_merged");

// Close (make sure tif is selected)
	close();							// activate me for batch
	
// Split channels
	run("Split Channels");


// 3) Create a mask on desired channel (called "DAPI") with Triangle threshold method + create ROI(s)
// Select window with DAPI channel ("DAPI" user defined in the beginning by dialogue)
selectWindow("C" + DAPI + "-" + "MAX_" + title);

// Run Threshold... with Triangle method. Feel free to change method below to get more fitting results.
// TROUBLE SHOOT option: Modifying threshold parameters by reducing the minimum size and minimum circularity (high circularity choosen for round opbjects)
setAutoThreshold("Triangle dark");

// Create a binary mask and modify mask to get outline of object without holes
// TROUBLE SHOOT option: If you have a too sensitive mask, try to run "dilate..." and "erode..." function a few times before adding to ROI manager.
run("Create Mask");
run("Dilate");
run("Fill Holes");
run("Watershed");

// Place ROI(s) on the outline of the mask(s) by run Analyze Particles... funciton and add to ROI manager. 
run("Analyze Particles...", "size=8000-100000 circularity=0.5-1.00 exclude add");																// Change here parameters to adjust!!!

// Goal: you got ROIs fitting to your DAPI signal to work with.


// 4) Reduce the ROI to 75% and place a new ROI in the center of detected object(s)
// Unselect and select ROI(s) 
// TROUBLE SHOOT option: Be extra carefull when changing here! Double check with recorder to be sure.
// NOT DONE yet: Note: in case no ROI(s) detected, window in the end will pop up saying "Attention: title no identified objects!"
// Alternatively: Run the function run("Fit Circle");
// Scale ROI(s) to 75% of their size. // Feel free to change the value 0.75 here to get an other scaling parameter.
// Add new ROI(s) in ROI manager.
// Update ROI manager by replacing previous ROI(s) with scaled ROI(s) 
count = roiManager("count");
current = roiManager("index");
for (i = 0; i < count; i++) {
	roiManager("select", i);
	run("Scale... ", "x=0.75 y=0.75 centered");
	roiManager("update");
}
if (current < 0)
	roiManager("deselect");
else
	roiManager("select", current);

// 5) Overlay selected ROI(s) to channel of interest (called "ChOI")
// Select channel of interest ("ChOI" user definied)
selectWindow("C" + ChOI + "-" + "MAX_" + title);

// Deselect and select all newly scaled ROI(s)
// Overlay ROI(s) on selected window with channel of interest window ("Show all").
roiManager("Show None");
roiManager("Show All");


// 6) Measure the previously set parameters within 75% object(s) + create results table
// Measure values of 75% reduced ROI(s) on channel of interest
// Result table shows the measured parameters (Set measurements...),
// each row represents one detected OBJECT (not one image). 
// Name: "C..." indicating measured channel number, "title of series", "0001..." number of object
// In case there is no detected object in an image, it will NOT show up in the table, but there will be a pop-up as well as the Log-print will tell
roiManager("Measure");


// 7) Save tifs of all images as merged channels and overlayed ROI(s) on channel of interest and mask  
// Add scale bar // Feel free to change predifined parameters
run("Scale Bar...", "width=100 height=6 font=24 color=White background=None location=[Lower Right] bold overlay");
// NOT DONE yet: Adjust channel by channel with previously set values
// Select window with channel of interest image // we assume it is still selected.
// Convert channel of interest image with ROI(s) to RGB format with Flatten function
run("Flatten");
// Save as tif with suffix _ROI in output directory
saving_title = replace(new_title, "/", "_" );
saveAs("Tiff", outputDir + saving_title + "_ROI");

// Select window called mask
selectWindow("mask");
// Convert mask image with ROI(s) to RGB format with Flatten function
run("Flatten");
// Save as tif renaming with title and suffix _mask in output directory
saving_title = replace(new_title, "/", "_" );
saveAs("Tiff", outputDir + saving_title + "_mask");
// Reset ROI manager (deletes ROI(s))
roiManager("reset");
// Close close close close                           // activate me for batch mode


run("Close All");



// 8) Export measurements as excel and as txt files
// "Saving to..." Log print
print("Saving to " + outputDir);

// End of the batch script					// activate for batch
			}
			
		} else if (endsWith(fileList[file], "/")) {
			processBioFormatFiles(currentDirectory + fileList[file]);
		}
	}
}

// Popup: You have successfully analyzed your data. NOT DONE - Attention: Images "title, title" had no detected objects.
// DONE. 


// If you have any suggestions, feel free to contact.
// TROUBLE SHOOT options show you most likely possibilities why the script does not work properly on your files
// Try to change the suggested parts and run it again